Overcoming drug resistance in multi-drug resistant cancers and microorganisms

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Bioengineered 3:5, 262–270; September/October 2012; © 2012 Landes Bioscience

262 Bioengineered volume 3 issue 5

*Correspondence to: Ananda M. Chakrabarty; Email: [email protected]: 05/16/12; Revised: 06/12/12; Accepted: 06/14/12http://dx.doi.org/10.4161/bioe.21130

resistance development against multiple drugs is a common feature among many pathogens—including bacteria such as Pseudomonas aeruginosa, viruses and parasites—and also among cancers. The reasons are 2-fold. Most commonly-used rationally-designed small molecule drugs or monoclonal antibodies, as well as antibiotics, strongly inhibit a key single step in the growth and proliferation of the pathogen or cancer cells. The disease agents quickly change or switch off this single target, or activate the efflux mechanisms to pump out the drug, thereby becoming resistant to the drug. A second problem is the way drugs are designed. The pharmaceutical industry chooses to use, by high-throughput screening, compounds that are maximally inhibitory to the key single step in the growth of the pathogen or cancer, thereby promoting selective pressure. An ideal drug would be one that inhibits multiple steps in the disease progression pathways with less stringency in these steps. Low levels of inhibition at multiple steps provide cumulative strong inhibitory effect, but little incentives or ability on the part of the pathogen/cancer to develop resistance. Such intelligent drug design involving multiple less stringent inhibitory steps is beyond the scope of the drug industry and requires evolutionary wisdom commonly possessed by bacteria. This review surveys assessments of the current clinical situation with regard to drug resistance in P. aeruginosa, and examines tools currently employed to limit this trend. we then provide a conceptual framework in which we explore the similarities between multi-drug resistance in pathogens and in cancers. we summarize promising work on anti-cancer drugs derived from the evolutionary wisdom of bacteria such as P. aeruginosa, and how such strategies can be the basis for how to look for candidate protein/peptide antibiotic drugs from bioengineered bugs. Such multi-domain proteins, unlike diffusible antibiotics, are not diffusible because of their large size and are often released only on contact with the perceived competitor. Thus, multi-domain proteins are missed during traditional methods of looking for growth zone inhibition of susceptible bacteria as demonstrated by antibiotics, but may represent the weapons of the future in the fights against both drug-resistant cancers and pathogens such as P. aeruginosa.

Overcoming drug resistance in multi-drug resistant cancers and microorganisms

A conceptual frameworkBenjamin S. Avner,1 Arsenio M. Fialho2 and Ananda M. Chakrabarty3,*

1Department of Physiology and Biophysics; University of illinois College of Medicine; Chicago, iL USA; 2institute for Biotechnology and Bioengineering; Department of Bioengineering; instituto Superior Tecnico; Lisbon, Portugal; 3Department of Microbiology and immunology; University of illinois College of Medicine; Chicago, iL USA

Key words: Pseudomonas aeruginosa, anti-pseudomonal drugs, multi-drug resistance, azurin, new-generation protein/peptide drugs

Introduction

Drug resistance in microorganisms,1 viruses2 and parasites3 is rampant and has created major problems in health care deliv-ery in the United States and indeed worldwide. Such resistance development is not only confined to drugs but also to neutraliz-ing antibodies, making it hard for effective vaccine development against bacteria such as Mycobacterium tuberculosis, viruses such as HIV-1, or parasites such as the malarial parasite Plasmodium falciparum. This concern applies to bacteria and other pathogens, but also analogously to the neoplastic cell populations which cause cancers.4 The situation is particularly difficult with bacteria such as multi-drug-resistant (MDR) or extensively-drug-resistant M. tuberculosis, methicillin, vancomycin-resistant Staphylococci, and multi-drug-resistant Pseudomonas aeruginosa. The potential crisis has captured attention on the political stage, as exempli-fied by the GAIN (Generating Antibiotic Incentives Now) bill introduced to the United States Senate. Much of this resistance development has been ascribed to the indiscriminate overuse of the antibiotics in areas other than medicine such as in the meat industry by incorporating antibiotics in the feed stocks to prevent infections in domestic or feedlot animals. Certainly, the high rates of mutations in genes conferring resistance such as the genes encoding the ABC transporter efflux pumps, and their associa-tion with transmissible plasmids1 for their rapid dissemination, have had an enormous impact on the increasing lack of efficacy of antibiotics in treating various life-threatening infections. In this review, we summarize the current clinical picture of increasing antibiotic resistance in MDR P. aeruginosa, and the limitations of antibiotic therapy. We include a short discussion of measures currently being undertaken to maximize the value of currently available therapeutic options. It is noteworthy that since antibiot-ics or small molecule anti-cancer drugs often target a single step in the disease progression pathway, any disease agent may quickly become drug-resistant with the correct mutation. We therefore subsequently introduce the concept that bacterial protein/pep-tide drugs with multiple domains, rather than small molecule compounds similar to antibiotics, may represent a superior strat-egy to inhibit key steps in the disease progression pathways of cancers and bacterial infections alike. Indeed, the pseudomonal redox protein azurin appears to act as a weapon against multiple

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www.landesbioscience.com Bioengineered 263

do have anti-pseudomonal activity. Piperacillin-tazobactam (PIP-TAZ) in particular remains a drug combination in very widespread use as a broad-spectrum agent with relatively high effec-tiveness. By many analyses, PIP-TAZ is the most effective non-polymyxin anti-pseudomonal drug in current use, although questions exist about whether the current thresholds for “susceptibil-ity” are appropriate.9 Other β-lactams useful against pseudomonal infections include certain new-generation cephalosporins, most notably ceftazidime and cefepime. The carbapenems (i.e., imipenem and meropenem) are broad-spectrum β-lactams with β-lactamase resistance which have long been viewed as the drugs of choice for Gram-negative organisms resistant to other drugs; with the exception of ertapenem, drugs in this class have strong anti-pseudomonal activity. In association with more frequent use, carbapenem resistance is now increasing among Gram-negative organisms.10

Anti-pseudomonal drugs from other anti-biotic classes include fluroquinolones such as ciprofloxacin and levofloxacin, broad-spectrum

antibiotics commonly employed not only in the ICU but also throughout both the inpatient and the community settings. These drugs can also be orally dosed and have generally favorable safety profiles, further contributing to their popularity. As dis-cussed in greater detail below, fluoroquinolone resistant P. aeru-ginosa is now extremely common, leading some to conclude that “fluoroquinolones are no longer adequate for empiric therapy of infections caused by P. aeruginosa.”11 The aminoglycosides (ami-kacin, gentamicin, tobramycin) are highly bacteriocidal antibi-otics whose utility is limited by their nephro- and oto-toxicity. In the past decade, polymyxins such as colistin (polymyxin E) and polymyxin B have re-entered extensive clinical use due to the emergence of MDR P. aeruginosa. The polymyxins also carry significant concern about toxicity, particularly nephro- and neuro-toxicity.

Antibiotic Resistance in P. aerugniosa

P. aeruginosa is intrinsically resistant to many anti-microbial drugs and rapidly acquires immunity to others. Additionally, “sequential resistance,” in which an isolate already resistant to one drug is then treated with another, is a likely means for the creation of MDR P. aeruginosa.12 As a result, MDR Pseudomonas are isolated worldwide. Recent statistics show that the resistance of P. aeruginosa isolates to different classes of antibiotics var-ies greatly depending on the country, ranging from 45% to less than 1% (Fig. 1), particularly when in-dwelling medical devices are used triggering biofilm formation (Fig. 2). Furthermore, multiple studies have demonstrated a correlation between past antibiotic exposure and infection with bacteria resistant to that antibiotic.8,11,13 Riou et al.8 and others further demonstrated that in an acute setting, the resistance of nosocomial P. aeruginosa

invaders—infectious and malignant—via multiple mechanisms of action. Even as P. aeruginosa itself produces factors which have great potential as anti-neoplastic agents, similar therapeutic development strategies are indicated to address the current situa-tion with multi-drug-resistant strains of P. aeruginosa.

Pseudomonas aeruginosa: The Current Clinical Picture

Pseudomonas aeruginosa is an aerobic Gram-negative bacillus, growing primarily in soil but also able to survive in the human body as an extracellular producer of bio-films. It is known primar-ily as a nosocomial pathogen, capable of infection at all sites of the body. P. aeruginosa is particularly prevalent in intensive care units (ICUs).5 The bacterium appears to be highly adapted to the respiratory tract, and is infamous as the most common respira-tory pathogen in patients with cystic fibrosis (CF).6 Numerous other clinical infections attributable to this bacterium include bacteremia, bone and joint infection, meningitis, eye infection, otitis externa [including “swimmer’s ear” in children and necro-tizing (“malignant”) otits externa], burn and wound infections, and urinary tract infections.6,7 Estimates from samples collected by the Centers for Disease Control and Prevention in the 1980s and 1990s indicate that P. aeruginosa is the second most common pathogen responsible for nosocomial pneumonia.5 The preva-lence of this organism in a nosocomial setting is matched by its deadliness, with P. aeruginosa pneumonia carrying a mortality rate of 30–40%.8

Available anti-pseudomonal drugs include β-lactam drugs and antibiotics from other classes. Although most traditional penicillins are ineffective against Pseudomonas species, certain new-generation penicillins combined with β-lactamase inhibitors

Figure 1. Percentage of antibiotic-resistant Pseudomonas aeruginosa to various classes of antibiotics across selected countries (2009). The data were collected from CDDeP, The Center for Disease Dynamics, economics and Policy (http://www.cddep.org/).

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PIP-TAZ also was effective in a significantly lower proportion of the 1999 isolates compared with the 1997–8; fluoroquinolones as a group also demonstrated significantly reduced effectiveness. The authors documented that no anti-pseudomonal was effec-tive against more than 90% of the isolates obtained. The wide geographic variation found in this study may be attributable to sampling variability, differences in native resistance patterns, or differences in antibiotic treatment practices in different regions. These early studies document clearly the potential for resistant strains to spread worldwide. It is particularly noteworthy that no antibiotic exists to which resistant strains have not also emerged. Contrary to previous belief that polymyxins, as a drug class, did not promote stable antibiotic resistance,12 examples of polymyxin resistance are now reported.8,15

The exact rate at which this potential epidemic is progress-ing is not entirely clear, particularly in light of findings from the mid-2000s suggesting that resistance to fluoroquiniolones and other drugs might have reached a plateau despite a continued increase in PIP-TAZ resistant and MDR P. aeruginosa.7 The pre-viously mentioned report by Riou et al.8 utilizing samples from multiple Belgian hospitals paints a particularly grim picture. The

pneumonia to multiple anti-pseudomonal antibiotics was higher at the end of an ICU course when compared with the initial culture.

Emerging resistance to fluoroquinolones is particularly com-mon (Table 1). Worldwide, even the most potent member of the class, ciprofloxacin, was effective against only 60–75% of clinical isolates by 1999.5 Neuhauser et al. surveyed isolates from ICUs throughout the United States and found a decline in ciprofloxa-cin susceptibility from 89% to 63% of P. aeruginosa isolates between 1993 and 2000. As these authors point out, this period of time is also notable for a marked increase in the use of cip-rofloxacin and other fluroquinolones. Other estimates of fluro-quinolone resistance in the ICU suggest rates at above 30% by 2002, doubled compare with a decade earlier.11 Similarly, P. aeru-ginosa isolates from “clinically significant” infections as part of the Global SENTRY Antimicrobial Surveillance Program5 col-lected between 1997 and 1999 demonstrated a general decline in the proportion of isolates that were antibiotic-susceptible. These authors reported a statistically significant increase in resistance to nearly all anti-pseudomonal antibiotics in Europe (i.e., PIP-TAZ susceptibility declined from 90% to 74%). In the United States,

Figure 2. Percentage of antibiotic-resistant Pseudomonas aeruginosa associated with device-related infections (January 2006–October 2007). These data were extracted from Hidron et al.42 CPM, cefepime; TAZ, ceftazidime; iMi, imipenem; MerO, meropenem; AMK, amikacin; PiP, piperacillin; PTZ, piperacillin-tazobactam; FQs, Fluoroquinolones.

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It is notable that the antimicrobial class that consistently con-tinues to engender high and growing resistance is the fluoroqui-nolones, the only class of anti-pseudomonals that can be widely used in the outpatient community setting, raising the question of whether community antibiotic prescribing practices may be as important or more so than hospital practices. Karlowsky et al.7 also note that their data regarding apparent stabilization of cip-rofloxacin resistance rates were collected at a time (2001–2003) shortly after a widespread movement among clinicians to phase out ciprofloxacin for P. aeruginoas treatment in favor of other drugs such as levofloxacin.

The existence of MDR P. aeruginosa leads to the intuitive conclusion that increasing resistance will lead to an increase in morbidity and mortality, as suggested by several case series16 and retrospective studies.17 An attempt to quantify the clinical impact of multi-drug resistance via literature review concluded that most studies report an increase in mortality attributable to MDR P. aeruginosa, but that differences in definitions and methodolog-ical limitations make the data difficult to compare or analyze.10 It should be noted that few trials exist directly comparing sus-ceptible and resistant organisms. However, conducting a retro-spective analysis in which patients whose cultures demonstrated the presence of levofloxacin susceptible P. aeruginosa as compared

isolates for this study were clinical isolates from various body sites of ICU patients diagnosed with hospital-acquired pneumo-nia. Although the study was not designed as an epidemiological estimate, the authors report a greater than 25% resistance among these infections to PIP-TAZO, cefipime and others, worsening over the course of the ICU stay. More than half of these isolates showed some degree of resistance to polymyxins. The authors’ conclusion is that the results “clearly show that the clinician’s choice of active antibiotics has become increasingly narrow.” On the other hand, Master et al.9 analyzed mixed colonization and infection data from The Surveillance Network Database. The data indicate increasing resistance to ciprofloxacin among P. aeruginosa consistent with prior reports, as well as a smaller effect on gentamicin. However, they also report some surprising findings, including a slight “recovery” of ciprofloxacin effective-ness after 2004, relative stability of susceptibility rates to most other antibiotics, and a small but statistically significant decrease in the percentage of isolates that were resistant to four or more drugs in 2009 compared with 2000. The authors suggest that perhaps infection-control measures and antimicrobial steward-ship programs as discussed below are effective. We summarize published assessments of prevalence and change of fluoroquino-lone and other antibiotic resistance by P. aeruginosa in Table 1.

Table 1. Studies assessing percentage of P. aeruginosa with antibiotic resistance

Total n SampleYear(s) studied

Initial ciprofloxacin resistance

Final ciprofloxacin resistance

Other Δ

Gasink et al. 200611 4976

P. aeruginosa isolates from US hospital

1991–2000 15% 41% (levofloxacin)

Neuhauser et al. 200314 8244

Consecutive isolates from iCUs in 43 states

1994–2000 11% 32%

Gales et al. 20015 6631

Clinical infections from international network of

sentinel hospitals1997–1999 20.2% USA 24.6% USA

· Piperacillin/tazobactam USA:10.1 to 13.4% europe: 9.9

to 26.2%

· Cefepime USA:22.3 to 16.9%; europe:19.2 to 25.2%

· imipenem USA: Δ non-signifi-cant; europe: 11.7 to 28.4%

Master et al. 20119 924,740

Clinical infections from 25 hospitals

1997–2009 20% 25%resistance to any drug

(2000–2009): 59.2 to 55.8%

Livermore CiD 200212 (data

from The Surveillance

Network Database USA)

2,194Clinical infections from 25

hospitals1997–2000 29.5% MDr isolates: 12.8 to 20.8%

Karlowsky et al. 20057 > 2,300

Consecutive Pseudomonal specimens

from 27 US hospitals2001–2003 33.5% 31.2%

· imipenem: 15.6 to 21.2%

· MDr isolates: 7.2 to 9.9%

Bennett et al. 200724

106 SiCU, 68 MiCU

Clinical infections from single hospital, MiCU and SiCU recorded separately

2002–2004SiCU: 29%

MiCU: 50%

SiCU:35% (Δ N.S.)

MiCU: 72% (Δ N.S.)

riou et al. 20108 110

Clinical isolates from pts with nosocomial

pneumonia, 5 Belgian hospitals

2006–2009

20%

PiP-TAZ, resistance: > 25%

cephalosporin resis-tance: > 25%

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drug with an aminoglycoside, despite the fact that its therapeutic value is unclear. Although susceptibility testing generally sug-gests that combination therapy is superior to a β-lactam alone,7 a meta-analysis of 64 randomized trials21 concluded that adding an aminoglycoside provided no clinical benefit in all-cause mor-tality in patients with severe infection compared with β-lactam mono-therapy. These findings support the general notion that appropriate mono-therapy based on susceptibility data are clini-cally equivalent to combination therapy.20 In vitro studies of levofloxacin/imipenem co-administration on different strains of P. aeruginosa over a 30-h period indicated that this combination was generally superior to either drug alone, even in strains that were partially or fully resistant to one of the two agents.22 To our knowledge, this effect has not been further investigated in a patient setting. Other investigators propose novel antibiotic com-binations incorporating drugs not traditionally thought to have anti-pseudomonal activity, such as rifampin, whose potential mechanism is unknown.20 It should also be noted that regard-less of the ultimate treatment once cultures are grown and sus-ceptibility data obtained, the practice of combination therapy will remain widely used by clinicians who feel that, during the empiric stage of treating a clinical infection (prior to culture growth), combination therapy may limit the mortality associ-ated with inappropriate initial drug selection.19 For instance, the guidelines for empiric therapy for health-care-associated pneu-monia by the American Thoracic Society and the Infectious Diseases Society of America (Guidelines 2005) do recommend using multiple anti-pseudomonal agents, i.e., a β-lactam plus a fluoroquinolone or aminoglycoside. As of the time of this writ-ing, the most recent version of these guidelines was published in 2005, with an update scheduled for late 2012. A full evaluation of combination therapy is beyond the scope of this review, but any consensus appears to be far away on the question of whether and under what circumstances it is appropriate for the treatment of P. aeruginosa infection.

Antimicrobial cycling. Regular cycling of empiric therapy for presumed Gram-negative infections, such that each individual drug class spends frequent time in limited use, provides the poten-tial to transiently reduce selective pressure and prolong a drug’s effectiveness. Some early studies employing antibiotic cycling strategies to limit ventilator-associated pneumonia (VAP) caused by Gram-negative bacteria yielded promising results, although methodological limitations have been cited—most notably lack of true control groups.19 Nijssen et al.,23 focusing on ICU-based studies of antimicrobial cycling, identified numerous issues with most old and new research on the topic. The most common study design is a “before-after” model in which the “control” and “inter-vention” arms of the study are temporally separated. Obviously, this introduces tremendous potential for confounding variables. Specifically: (1) Resistance patterns in ICUs normally exhibit baseline variation over time; (2) Resistance patterns in the sur-rounding community may change over the course of a study; (3) Hospital infection control measures may change over the course of a study. Other concerns identified by Nijssen et al. include the difficulty of comparing studies of colonization (incorporating surveillance cultures) with those that limit their focus to clinical

with those with levofoxacin-resistant P. aeruginosa, Gasink et al.11

found higher hospital mortality and economic burden caused by resistant isolates; in multi-variate analysis, carbapenem suscepti-bility rather than fluoroquinolone susceptibility was significantly associated with mortality, possibly due to a delay in initiating appropriate therapy.

The Antibiotic Shortage and Strategies for the Near Future

Only one new anti-pseudomonal drug had been approved in recent years (doripenem), and as of 2009, no new anti-pseudo-monal drugs were undergoing phase 3 clinical trials7,10—a situ-ation which limits treatment options in the foreseeable future given the length of time required for clinical drug development. The Infectious Diseases Society of America (IDSA) has lamented the decline in rate of introduction of new anti-bacterials and drug companies’ “decreased investment in this therapeutic area.”18 The results of a literature and database search also led the IDSA to conclude that drugs currently in the developmental pipeline will not address the emergence of carbapenem-resistant Gram-negative bacteria, and to express concern over the paucity of com-pounds with novel mechanisms of action.

As the authors of the above report point out, multiple factors limit pharmaceutical companies’ investment in anti-microbials, including the acute nature of the conditions treated and per-ceived regulatory hurdles (large necessary trial size, unfeasibility of placebo-controlled trials for ethical reasons). The IDSA advo-cates for federal action to provide incentives to encourage the development of new antibiotics. Indeed, at the time of this writ-ing, the United States Senate is considering a bill, introduced by Senator Richard Blumenthal, D-Conn and Senator Bob Corker, R-Tenn, called GAIN (Generating Antibiotic Incentives Now), that would provide drug-makers 10 y of exclusive marketing rights and would allow the US Food and Drug Administration (FDA) to speedily approve such drugs without compromising the safety and efficacy issues, thereby minimizing regulatory complications.

Due to the concern that antibiotic resistance, particularly in the ICU, will outstrip the development of appropriate anti-microbial therapy, clinical utilization strategies of existing anti-microbials is an area of active investigation. Potential options include formal guidelines, optimized pharmacokinetics, and shorter courses of antibiotic treatment.19 We will now briefly dis-cuss three additional strategies: combination therapy, antibiotic “cycling” (in which drugs used for empiric treatment are rotated on a scheduled basis), and improvement in infection-control measures.

Combination therapy. Controversy currently exists about whether antibiotic resistance rates can be affected, favorably or unfavorably, by the practice of combination therapy—the use of multiple antibiotics rather than mono-therapy to treat known or suspected pseudomonal infections. Possible but unproven ben-efits include synergistic therapeutic effects as well as possible prevention of resistance developing to one drug or the other.20 One combination in common clinical use is that of a β-lactam

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ertapenem, a drug which may potentially limit the use of the anti-pseudomonal carbapenems), the hospital also implemented increased education promoting hand hygiene and supply of alco-hol-based hand rub. Hand rub consumption was correlated with the net trend of reduction in the resistant Pseudomonas (the data were statistically significant for the overall trend, not in direct comparisons of any two time periods), whereas the association with the stewardship intervention was not significant. The litera-ture reports a wide range of adherence to hand hygiene protocols, often very low.16,28,29 Perhaps for this reason, hand hygiene alone does not appear to be sufficient to prevent nosocomial infection, and in one case report,16 an ICU outbreak of MDR P. aerugi-nosa was only contained after the center instituted pasteurization of water taps. If infection control is to be successfully used to limit the emergence of antibiotic resistance, the combination of resources and education toward hand hygiene, personal protec-tive equipment, environmental sterilization, and patient cohort-ing are likely to all be essential.

A Long-Term Approach: Beyond Small Molecule Anti-Microbials

For the more distant future, the new drugs under development include both traditional antibiotics and antibody fragments to reduce pathogenicity.7 However, it is clear that the ability of P. aeruginosa to evolve resistance to anti-microbials will continue to possess the potential to outstrip drug development. The situa-tion exposes the fact that standard approaches to antibiotic ther-apy are inherently limited.

Bacterial “evolutionary wisdom” and the example of azurin. Antibiotics are generally small molecules produced by or pat-terned after products produced by slow-growing soil micro-organisms such as Actinomycetes in order to compete with faster-growing neighbors.30 When a soil-dwelling organism such as P. aeruginosa also lives in a setting such as the human body, it may also be drawn into competition with viruses, parasites, can-cers, and other bacterial pathogens, any of which may threaten to out-compete the bacteria or simply kill the host. It stands to reason that over three billion years of evolution, successful patho-genic bacteria will have developed the “evolutionary wisdom” to produce tools to compete with “enemies” within a eukaryotic host. Furthermore, weapons designed by evolutionary wisdom might be expected to include molecules with multiple domains to target the multiple invasive apparatuses. Due to the limited genome sizes of bacteria, an ideal weapon would also be able to attack multiple different enemies. In sum, just as evolutionary wisdom in soil-dwelling fungi produced the antibiotics currently in use, evolutionary wisdom in pathogenic bacteria may have also designed multi-domain products very different from commercial antibiotics, which generally have a single or limited number of target sites. In this respect, standard pharmaceutical approaches for antibiotic detection and in-hospital susceptibility testing are inadequate, relying on diffusion of small molecules in agar plates to produce zones of inhibition of susceptible bacteria or fungi. This detection of growth inhibition zones, which can only be exhibited by small molecules diffusing out of the producer

infection, the fact that the usual definition of nosocomial infec-tion (> 48 h after hospital admission) has not been thoroughly validated, and the fact that most studies do not directly measure levels of patient to patient transmission (this may distort data due to the fact that many standard statistical tests assume that each patient represents an independent data point).

One group of authors reports a comparison between antibio-grams of organisms isolated from clinical infections in a surgical ICU (SICU) and a medical ICU (MICU).24 The SICU imple-mented a monthly antibiotic cycling regimen, while the MICU did not. The authors do not describe any attempt to quantify the differences in demographics or clinical status between the two patient populations. Nonetheless, this represents one of the few evaluations of the cycling strategy which studied the “control” and “intervention” strategies concurrently. In this setting, suscep-tibility of P. aeruginosa was higher to both PIP-TAZ and ceftazi-dime after the intervention than before; this favorable change was observed only in the SICU, where cycling was employed, and not in the MICU. Not all results have supported cycling, particu-larly with longer cycle length. A pair of before-after studies using similar protocols25,26 conducted in ICUs in which the primary antibiotic was rotated on quarterly intervals were both unable to detect a reduction in P. aeruginosa antibiotic resistance per se. The authors of the Warren et al., study do note that patients appeared to be initially sicker and had longer ICU stays during the cycling period, and that their ICU did not show the same reduction of susceptibility observed in Gram-negative organisms elsewhere in the hospital. Hedrick et al. reported that a smaller percentage of ICU-acquired infections were fatal during the cycling period. At this site, fluroquinolones were removed from the rotation due to an outbreak of MDR P. aeruginosa during this cycle. These accu-mulated findings lead to the conclusions that general uncertainty exists regarding the value of anti-microbial cycling to limit resis-tance in P. aeruginosa, and that shorter rotation intervals (i.e., monthly) appear to have the highest likelihood of success.

Infection control measures. Research is also shifting toward attempts to quantify the value of reducing the infectious bur-den of MDR bacteria by targeting transmission—limiting the absolute number of infections and, as a result of this, limiting antibiotic use. One particularly promising result comes from a before-after study in a Thai hospital27 assessing the efficacy of multiple interventions: hand hygiene protocols, personal protec-tive equipment, environmental cleaning, and patient cohorting. The authors report lower incidence of MDR A. baumannii colo-nization and infection after the intervention. Interpretation is limited by the before-after study design, particularly given that this hospital was experiencing an upswing in MDR A. bauman-nii at the time of the non-intervention period. Nonetheless, such results provide support for the principle that such an intervention should be investigated as a potential means to limit the emergence of resistance in other Gram-negative bacteria such as P. aerugi-nosa. Another investigation28 considered several independent variables in a multiple regression analysis of a decrease in isolation of carbapenem-resistant P. aeruginosa. During the study period, concurrent interventions were undertaken. Besides an antimicro-bial stewardship measure (introduction and discontinuation of

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already harboring P. aeruginosa frequently show declines in P. aeruginosa titers, leaving Burkholderia as the predominant pathogen;38,39 combined Burkholderia and Pseudomonas infec-tions are rare. In our laboratory, we have shown that when a CF-isolate strain of B. cepacia was grown on an agar plate and cross-streaked with a CF-isolate of P. aeruginosa, there was no appreciable zone of growth inhibition, suggesting that the B. cepacia strain did not produce diffusible antibiotics against the P. aeruginosa strain. Yet, when the two bacteria were co-cul-tured, the B. cepacia strains predominated, presumably through production of growth inhibitory materials, as the B. cepacia strain did not show faster growth than the P. aeruginosa strain in the growth medium employed as mono-cultures. Screening of the growth media of secreted proteins, when the two bacte-ria were grown in mono-cultures and as mixed cultures, indeed showed the presence of several new protein bands in the co-culture growth media. Such results appear to indicate that cer-tain bacteria can limit the growth of other bacteria through weapons other than antibiotics—perhaps proteins that are only secreted when the producer bacterium is physically confronted with the competitor.

Conclusion

The rise of MDR P. aeruginosa represents a looming threat to patient health in the near future, conjuring fears of a return to pre-antibiotic morbidity and mortality levels for the many infections caused by P. aeruginosa. Evidence demonstrates the increasing ineffectiveness of both β-lactams and other non-β-lactams—particularly widely used antibiotics such as fluoroquinolones. Resistance of P. aeruginosa to all existing anti-pseudomonal drugs, including “new” agents such as polymyxins, is reported. This rate of decline in drug efficacy threatens to outstrip the discovery and approval of new anti-bacterial drugs, which have also been in decline over the past several decades. Strategies to maximize the lifetime of currently available drugs already in place in some clinical settings include combination therapy and antimicrobial cycling. However, in many cases, a paucity of clini-cal studies of these interventions and methodological limitations of those studies prevent us from drawing firm conclusions as to their effectiveness; more research is urgently needed. Improved disease-control measures are likely to prove essential in slowing or stopping the development of resistance to existing antibiot-ics, but the exact value of their contribution is also unknown.

microorganism, prevents the detection of larger molecules such as proteins. Certain bacteria employ proteins to inhibit the growth of competitors in the human body (prokaryotes, viruses, parasites or even cancer) when such a bacterium is resident in the human body in the state of extremely slow-growing biofilms.31,32 A sec-ond drawback of the current antibiotic isolation method is that it fails to detect compounds released by bacteria only when in physical contact with an enemy.32

One example of evolutionary wisdom as a potential source for novel defenses against threats to the human body comes in the form of the bacterial redox protein azurin, produced by bacteria such as P. aeruginosa.31 Azurin demonstrates the ability to inhibit cancer growth.32 A chemically synthesized 28 amino-acid peptide derived from the protein, termed p28, is currently undergoing clinical trials, including a recently completed phase I study con-ducted in individuals with end-stage (stage IV) metastatic can-cers refractory to all conventional treatment. Of the 15 patients treated with p28, stabilization of disease occurred in 6 patients, partial regression of tumor in 2 patients, and complete regres-sion of the tumors in 2 other patients, without demonstrating any toxicity or side effects even at the highest dose (Table 2).33 Besides its anti-neoplastic abilities, the azurin protein also inhib-its the growth of other infectious agents, including the malarial parasite Plasmodium falciparum, the toxoplasmosis-causing para-site Toxoplasma gondii and the human AIDS virus HIV-1.32 Our research has also uncovered other bacterial proteins and peptides with potential activity against pathogens and cancers.34,35 This is the case with MPT63, a 16 kDa protein from Mycobacterium (M. tuberculosis, M. bovis) which, like azurin, exhibits anti-can-cer and anti-HIV activity.36 Interestingly, it has previously been noted that multi-drug resistance actually emerges in bacteria by some mechanisms similar to processes observed in cancer cells, such as the overexpression of efflux pumps of the ABC superfam-ily.37 Bacterially-derived protein/peptide products thus appear to demonstrate modes of action against cancer and infectious pathogens that are unique compared with conventional drugs, including drug-resistant cancers (Table 2).

Applying evolutionary wisdom against MDR P. aeruginosa. Might bacterial evolutionary wisdom also provide us with useful compounds in the case of multiple bacterial pathogens competing for a similar niche? Such a situation may, in fact, be manifested in the case of Burkholderia spp (i.e., B. cepacia, B. cenocepacia, B. multivorans), another major cause of lung infection in patients with CF. When co-infected with Burkholderia spp, CF patients

Table 2. results of human phase i clinical trials of p28 in stage iv cancer patients with metastatic, refractory solid tumors

PatientsType of cancer

Treatment modalityDosage

(mg p28/kg)Adverse events Objective response

Number

11 male

15

4 female

Melanoma (7)

Colon (4)

Sarcoma (2)

Pancreatic (1)

Prostate (1)

i.v. bolus of p28

3 times per week for 4 weeks

0.83

1.66

2.5

3.33

4.16

in escalating doses

None attributable to p28 (grade 1)

Stable disease (6/15)

Partial tumor regression (2/15, 1 prostate, 1 melanoma)

Age range

Median

50–80

62

Observation 2 weeks before the next higher

dose

No immune response or adverse effect even at

maximum dosage of p28

Complete regression (2/15, 1 sarcoma, 1 melanoma)

Data taken from a presentation made at the ASCO meeting in Chicago on June 6, 2011.33

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It is also important to note that bacterial protein weapons such as azurin have structural similarity with human immunoglobulins, thereby rendering such proteins largely non-immunogenic and contributing to their stability in blood.31,32 Strategies guided by the evolutionary wisdom of pathogenic bacteria through produc-tion of smart protein weapons may provide important contribu-tions to keeping all of these invaders at bay, as such pathogens are unlikely to mutate quickly in multiple steps that are inhibited by a protein or peptide drug with low levels of stringency.

Acknowledgments

We thank Dr Jeremy Young for his helpful advice during an initial survey of published literature. Part of our approach to develop Mycobacterial protein/peptide drugs targeted against cancers is supported by the Department of Biotechnology (DBT), Government of India, under the BIPP program awarded to Amrita Therapeutics (www.amritatherapeutics.com). Part of the work related with new drugs from bacterial origin is based upon work supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal (grants PTDC/EBB-BIO/100326/2008 and PTDC/BIA-MIC/118386/2010).

In the years to come, genuinely novel approaches, as outlined briefly in this review, will continually be necessary in the fight against development of all multiply drug-resistant pathogens. Approaches based on small molecules will always be especially prone to development of anti-drug resistance, a problem which also represents a difficulty in treating cancers. In recent years, major advances have been made in the therapeutic intervention of cancers, including combination drug therapy, but the prog-ress has been stymied by rapid resistance development, mostly through overexpression of ABC transporter efflux pumps40 or other mechanisms. Indeed, the mode of multi drug resistance development through overexpression of efflux pumps may have some similarity in both bacteria and cancers,37 as noted in this review. It should be noted, however, that there are other param-eters besides drug resistance that dictate, at least to some extent, the stability of a drug in the body and its efficacy. Such efficacy is also dependent on the absorption, distribution, metabolism, excretion and toxicity (ADMET) of the drug. This in turn might be modulated by ABC transporters,41 thereby linking drug resis-tance development and drug efficacy to a common parameter that might be independent of the nature or the size of the drug.

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Overcoming drug resistance in multi-drug resistant cancers and microorganisms